F. tularensis is a Category A select agent that can cause severe and fatal disease in humans if an aerosol containing as few as 10-100 CPU is inhaled. Indeed, in years past several countries weaponized F. tularensis for use as a possible biological weapon. For these reasons, it is now considered a potential bioterror agent and is a priority for countermeasure development. One live vaccine strain LVS, based on a biotype B strain, was developed and tested in the 1950's and 1960's and conferred partial protection against exposure to aerosols containing wild type virulent Type A F. tularensis. While providing proof of principle that a live attenuated F. tularensis vaccine can protect, LVS is not licensed by the FDA and suffers from several notable drawbacks that render it a sub-optimal tularemia vaccine. An optimal live vaccine will be based on introducing precisely defined attenuating mutations into a virulent Type A parent strain and will confer strong protection against exposure to aerosolized virulent Type A F. tularensis. Within the current MARCE funding cycle, we developed genetic tools to produce specifically targeted mutations in Type A and B F. tularensis. Multiple separate loci were identified that when inactivated render the prototype Type A strain, SchuS4, avirulent in a mouse model. In this grant application we will complete the construction of a series of double mutant strains, each containing two independently attenuating mutations. Working in collaboration with Dr. Stefanie Vogel's lab the vaccine strains will be evaluated for induction of innate and adaptive responses following infection of macrophages, in order to downselect the most promising strains to advance to animal studies. The optimal vaccine candidates will progress through a series of systematic preclinical rabbit studies to assess their safety, immunogenicity and capacity to protect against a against wild type aerosol challenge with SchuS4 (in collaboration with investigators at UMB and the University of Pittsburgh RBL). Completion of these will result in the identification of a live attenuated Ft vaccine candidate(s) ready to advance to human clinical studies of safety and immunogencity and non human primate studies of efficacy against virulent challenge.